The morphokinetics algorithm based on data from day 5 blastocyst transfer (KIDScoreD5 version 3) is also useful for embryo selection in day 6 blastocyst transfer

Abstract Purpose To analyze whether the morphokinetics algorithm based on data from day 5 blastocyst transfer (KIDScoreD5 version 3) can predict the pregnancy rate of both day 5 and day 6 blastocyst transfers. Methods The relationship between KIDScoreD5 and clinical pregnancy rate was evaluated using the Cochran–Armitage test and receiver‐operating characteristic (ROC) curve analysis. Results A positive correlation was observed between the KIDScoreD5 value and clinical pregnancy rate for both day 5 (p = 0.0003) and day 6 blastocysts (p = 0.0019) using the Cochrane–Armitage test. ROC curve analysis showed that the area under the curve (AUC) of KIDScoreD5 for clinical pregnancy was 0.627 (0.575–0.677, p < 0.0001) for day 5 blastocysts and 0.685 (0.571–0.780, p = 0.0009) for day 6 blastocysts. The combined analysis of both day 5 and day 6 blastocysts also showed an AUC of 0.680 (0.636–0.720, p < 0.0001), suggesting that it is possible to select embryos that are more likely to result in pregnancy. Conclusions KIDScoreD5 could predict pregnancy not only in day 5 blastocysts but also in day 6 blastocysts. When both day 5 and day 6 blastocysts are vitrified, embryo selection by KIDScoreD5 is possible with a high prediction ability of pregnancy.

reliably selecting embryos that result in pregnancy. 5 Moreover, because morphological evaluation depends on the subjectivity of the observer, it is not consistent. 6 One of the challenges of ART is to establish a simple and noninvasive method of embryo evaluation. Time-lapse incubators allow for a noninvasive, nonsubjective, and detailed observation of the embryo developmental dynamics, and their usefulness has been reported. Embryos with abnormal cleavages, such as direct cleavage, are associated with a low pregnancy rate, 7,8 and embryo cleavage at the correct time is associated with transfer outcomes. 9,10 Observing the early stages of embryonic development may be useful for the prediction of pregnancy potential. In addition, the proper speed of development of blastocyst is also an important evaluation point.
Embryos that develop into blastocysts on day 5 after fertilization are associated with a higher pregnancy rate than those that develop on day 6. 11 Therefore, embryo selection based on morphokinetics (developmental dynamics and morphological evaluation) has been attempted and reported to improve clinical outcomes. 12,13 Previous reports have recommended the evaluation of such morphokinetics parameters using a time-lapse incubator for embryo selection. 5,14,15 In a monocentric ambispective study (prospective and retrospective) conducted by Boucret et al., abnormal embryo development detection by time-lapse incubation was shown to improve embryo transfer outcomes. 5 Embryoscope+ (Vitrolife, Gothenburg, Sweden), is a time-lapse incubator that takes 11 focal plane pictures of culture embryos every 10 min and contains an algorithm, KIDScoreD5, for evaluating embryo morphokinetics. This algorithm is an embryo evaluation algorithm, which utilizes information from a large data set of known implantation data, comprehensively evaluates the developmental dynamics of the embryo, and performs morphological evaluation at the time of blastocyst formation, calculated in the form of a KIDScoreD5 value. [16][17][18] KIDScoreD5 does not require daily observations for annotation. Embryos that have developed into blastocysts are semiautomatically calculated the time to each developmental stage and morphological grade by the guided annotation. After the observer agrees or makes slight modifications to each calculated parameter using guided annotation, the blastocysts are evaluated with a KIDScoreD5 value ranging from 1.0 to 9.9.
The first model of KIDScoreD5 (version 1) evaluated the fading time of the pronucleus, the timing of cleavage from zygotes toward the eight-cell stage (t2, t3, t4, t5, and t8), the formation times of the blastocoele and blastocyst (tsB and tB), and the morphological grades of the inner cell mass (ICM) and trophectoderm (TE). In version 2, the timing of cleavage to the five-cell stage (t2, t3, t4, and t5) and tB and TE morphology were evaluated. Both algorithms were formed using the data of 1100 transferred embryos. Reigner et al.
reported that although both models predicted pregnancy, the value of the predictive ability was the same as that of traditional morphological evaluation (area under the curve [AUC] value = 0.60). 16 The latest algorithm, version 3, was recently created using 5200 implanted embryos, and ICM, which was not considered in version 2, was added to the algorithm. A previous study demonstrated that the value of KIDScoreD5 (version 3) is a good predictor of pregnancy and live birth of transferred embryos 17 ; however, in that study, day 5 and day 6 blastocysts were combined in the analysis. The KIDScoreD5 algorithm was based on day 5 blastocyst transfer, so it is unclear whether the transfer outcome of day 6 blastocysts transfer is also involved. Since a certain number of day 6 blastocyst transfers are also clinically performed, KIDScoreD5 must also be useful in selecting day 6 blastocysts. Therefore, the present study analyzed whether KIDScoreD5 (version 3) predicts the pregnancy rate of both day 5 and day 6 blastocyst transfers.

| Study design and population
This study was approved by the institutional review board of Takahashi Women's Clinic (protocol No. TWC20-001). Consent was obtained in the form of an opt-out through our clinic website and a bulletin board.

| Ovarian stimulation
Detailed ovarian stimulation protocols have been previously reported. 19 All patients underwent ovarian stimulation based on serum anti-Müllerian hormone (AMH) levels and follicle-stimulating hormone (FSH) levels on day 3 of the menstrual cycle. Ovarian stimulation was performed using the mild stimulation protocol with clomiphene citrate (CC), gonadotropin-releasing hormone (GnRH) antagonist protocol, or GnRH agonist protocol. Human chorionic gonadotropin and/or GnRH agonist was administered to induce ovulation when the leading follicle diameter reached 18 mm.

| Embryo culture and evaluation
Embryo culture was performed using EmbryoScope+ (Vitrolife) with an EmbryoSlide+ culture dish (Vitrolife). After ICSI, oocytes were transferred into ONESTEP medium® (Naka Medical) droplets under OVOIL® (Vitrolife). We took 11 focal plane embryo images every 10 min to obtain the developmental dynamics of the embryos.
Embryos showing two pronuclei were regarded as normally fertilized embryos and were cultured until the blastocyst stage up to 144 h at 37.0°C, 6.0% CO 2 , and 5.0% O 2 concentration.

| Embryo vitrification and single Vitrified-Warmed blastocyst transfer
Blastocysts with more than 50% blastocoel formation on day 5 and day 6 were vitrified using the Vitrification Kit (Kitazato, Fuji, Japan) according to the manufacturer's protocol. In the spontaneous ovulatory cycles, endometrial thickness and ovulation were monitored in the patients using ultrasound. Estradiol (3 mg/day; Julina®, Bayer, Leverkusen, Germany) and dydrogesterone (30 mg/ day; Duphaston®, Mylan Inc., Canonsburg, PA, USA) were orally administered when the endometrial thickness reached ≥8 mm and ovulation was confirmed. In the hormone replacement cycles, estradiol (1-3 mg/day) was administered daily beginning on days 3-5 of the menstrual cycle. Dydrogesterone (30 mg/day) was administered in addition to estradiol when the endometrium was confirmed to be ≥8 mm. In both embryo transfer cycles, embryo transfer was performed 5-6 days after initiating dydrogesterone administration.

| Statistical analysis
Statistical analysis was performed using JMP Pro 15.00 (SAS Institute, Cary, NC, USA). Categorical data were expressed as percentages and analyzed using the Chi-square test or Fisher's exact test. Continuous data were expressed as mean and standard deviation and analyzed using the Wilcoxon rank-sum test (Table 1) (Table 2).
Multivariate logistic regression analysis using confounding variables (female age at blastocyst vitrified, body mass index, basal AMH level, and etiology of infertility) was used to analyze the relationship between KIDScoreD5 value and clinical pregnancy (Table 3). In the analysis of day 6 blastocysts, the number of transfer cycles was limited; therefore, a stepwise variable selection procedure was used to include variables with the best Akaike information criterion. To evaluate the predictive ability for a clinical pregnancy, a receiveroperating characteristic (ROC) curve analysis with the KIDScoreD5 value and evaluated via AUC (Figure 3).    We analyzed the relationship between the clinical pregnancy rate and each 1.0 value of KIDScoreD5 using the Cochran-Armitage test (Table 2). A positive correlation was observed between KIDScoreD5 values and the pregnancy rate in the combined and separated analysis of day 5 and day 6 blastocysts.

| Relationship between KIDScoreD5 values and clinical pregnancy rate
Multivariate analysis showed that the KIDScoreD5 value affected the pregnancy outcome even when patient backgrounds and etiology of infertility were considered (Table 3).

| Evaluation of KIDScoreD5 and clinical pregnancy using an ROC curve analysis
To determine whether KIDScoreD5 is accurate for predicting pregnancy, ROC curve analysis with KIDScoreD5 was performed These results indicate that KIDScoreD5 can predict clinical pregnancy in both day 5 and day 6 blastocysts.   In recent years, the widespread use of time-lapse incubators has led to attempts to identify factors associated with pregnancy.

F I G U R E 2 Comparison of the mean
Developmental kinetics analysis is expected to further improve the prediction of pregnancy rate in the future. Sciorio et al. used the EmbryoScope drawing tool to measure the diameter and maximum area of blastocysts and analyzed their involvement in pregnancy rates, and found that the median diameter of nonclinical pregnant blastocysts was significantly lower than that of clinical pregnant blastocysts. 25 Furthermore, an increase in blastocyst maximum area of 1 μm resulted in a 2.6% increase in the odds ratio for pregnancy. 25 Therefore, expansion of the blastocyst area is likely to be a significant predictor of pregnancy. Another study reported that contraction of the blastocyst has a negative effect on transfer outcomes. 26 Blastocyst contraction and expansion is considered to cause thinning of the zona pellucida and promote hatching. However, a study by Niimura observed the amount of contraction in mouse embryos and reported that the hatching rate of embryos with strong contraction was lower than that of embryos with weak contraction. 27 Later, another report concluded that the evaluation of embryo contraction was a factor that predicted the outcome of pregnancy in human embryos regardless of the morphological quality of the blastocyst. 28 Because there has been a report of a higher number of contractions in aneuploid embryos, 29  and pregnancy may help in designing a more accurate pregnancy prediction algorithm.
The limitation of this study was that it was a retrospective analysis conducted in a single IVF center. There are differences in culture methods among IVF centers, and it is expected that the AUC of KIDScoreD5 will differ among facilities. In addition, the data used in this study were obtained from the analysis of 662 blastocyst transfers, and only 181 blastocysts were included in the analysis of day 6 blastocysts. Because of the limited number of blastocysts used in this study, the number of calculations should be increased, and the AUC values may change if studies are conducted at other centers. Therefore, randomized controlled trials at multiple centers are required.
In conclusion, the findings of this study showed that the embryo evaluation algorithm constructed from the results of day 5 blastocyst transfer also had a high predictive ability for pregnancy in day 6 blastocyst transfer. Even among patients with both vitrified day 5 and day 6 blastocysts, KIDScoreD5 may assist in selecting embryos that are more likely to result in pregnancy.

ACK N OWLED G M ENTS
We thank the staff of Takahashi Women's Clinic. The authors would like to thank Enago (www.enago.jp) for the English language review.

CO N FLI C T O F I NTE R E S T
Masashi Shioya, Tatsuya Kobayashi, Tomoharu Sugiura, Maki Fujita, and Keiichi Takahashi declare that they have no conflicts of interest.

E TH I C A L A PPROVA L
This study was approved by the institutional review board of Takahashi Women's Clinic (protocol number: TWC2-001).

I N FO R M E D CO N S E NT
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for their inclusion in the study.

H U M A N/A N I M A L R I G HTS
This article does not contain any experimental studies with human or animal participants on the part of any of the authors.